Earl E. Clarke

Involvement of Caspases in Proteolytic Cleavage of Alzheimer’s Amyloid-β Precursor Protein and Amyloidogenic Aβ Peptide Formation

Abstract The amyloid-β precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-β (Aβ) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimers disease brains and colocalization of its APP cleavage product with Aβ in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for Aβ peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimers disease.

Selected Non-steroidal Anti-inflammatory Drugs and Their Derivatives Target γ-Secretase at a Novel Site EVIDENCE FOR AN ALLOSTERIC MECHANISM

γ-Secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-β (Aβ) peptides from processing intermediates of the β-amyloid precursor protein. Because Aβ peptides are thought to be causative for Alzheimers disease, inhibiting γ-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of γ-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Aβ(1–42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Aβ(1–42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective γ-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the γ-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of γ (42)-cleavage over alternative cleavages in the β-amyloid precursor protein and, more importantly, alternative substrates.

A new series of potent benzodiazepine γ-secretase inhibitors

Abstract A new series of benzodiazepine-containing γ-secretase inhibitors with potential use in the treatment of Alzheimers disease is disclosed. Structure–activity relationships of the pendant hydrocinnamate side-chain which led to the preparation of highly potent inhibitors are described.

Quantitation of amyloid-β peptides in biological milieu using a novel homogeneous time-resolved fluorescence (HTRF) assay

Many of the recent advances in the understanding of the pathological processes underlying Alzheimers disease have come about as a result of the development of assays that can specifically quantitate in biological milieu amyloid-beta (A beta) peptides ending at amino-acid positions Ala-42 (A beta(42)) and Val-40 (A beta(40)). The existing technologies, however, although proven in their utility are limited in their application with regards to sample manipulation and suitability for high-throughput screening. To overcome these limitations, in this report we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) immunoassay for A beta(42) and A beta(40) peptides. This assay has the sensitivity, selectivity and dynamic range to allow specific, direct quantitation of A beta peptides in cell culture medium, plasma, cerebrospinal fluid and brain tissue extracts, and has the major advantage of minimising sample manipulation and its inherent inaccuracies.

Intra- or Intercomplex Binding to the γ-Secretase Enzyme A MODEL TO DIFFERENTIATE INHIBITOR CLASSES

γ-Secretase is one of the critical enzymes required for the generation of amyloid-β peptides from the β-amyloid precursor protein. Because amyloid-β peptides are generally accepted to play a key role in Alzheimer disease, γ-secretase inhibition holds the promise for a disease-modifying therapy for this neurodegenerative condition. Although recent progress has enhanced the understanding of the biology and composition of the γ-secretase enzyme complex, less information is available on the actual interaction of various inhibitor classes with the enzyme. Here we show that the two principal classes of inhibitor described in the scientific and patent literature, aspartyl protease transition state analogue and small molecule non-transition state inhibitors, display fundamental differences in the way they interact with the enzyme. Taking advantage of a γ-secretase enzyme overexpressing cellular system and different radiolabeled γ-secretase inhibitors, we observed that the maximal binding of non-transition state γ-secretase inhibitors accounts only for half the number of catalytic sites of the recombinant enzyme complex. This characteristic stoichiometry can be best accommodated with a model whereby the non-transition state inhibitors bind to a unique site at the interface of a dimeric enzyme. Subsequent competition studies confirm that this site appears to be targeted by the main classes of small molecule γ-secretase inhibitor. In contrast, the non-steroidal anti-inflammatory drug γ-secretase modulator sulindac sulfide displayed noncompetitive antagonism for all types of inhibitor. This finding suggests that non-steroidal anti-inflammatory drug-type γ-secretase modulators target an alternative site on the enzyme, thereby changing the conformation of the binding sites for γ-secretase inhibitors.

Functional Overexpression of γ-Secretase Reveals Protease-independent Trafficking Functions and a Critical Role of Lipids for Protease Activity

Presenilins appear to form the active center of γ-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue γ-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, γ-secretase appears to play a role in the trafficking of the β-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized γ-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. γ-Secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the γ-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive γ-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of γ-secretase.

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding

γ‐Secretase performs the final processing step in the generation of amyloid‐β (Aβ) peptides, which are believed to be causative for Alzheimers disease. Presenilins (PS) are required for γ‐secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen‐bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue γ‐secretase inhibitor. Here we report that replacement of either tyrosine residue alters γ‐secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Aβ(42) peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.